Kirkpatrick, D.S. et al. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nat. Cell Biol. 8, 700-710
Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts, United States Nature Cell Biology
(Impact Factor: 19.68).
08/2006; 8(7):700-10. DOI: 10.1038/ncb1436
Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.
Available from: Serena Faggiano
- "Cation exchange chromatography on a MonoS column (GE Life Sciences) is used for the purification of K33-linked chains. A quantitative mass spectrometry method (AQUA)  is used to assess the purity of the product . The same technique has been used previously also for the assessment of the K11-, K48-, and K63-linked chains . "
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ABSTRACT: Attachment of ubiquitin (Ub) as mono- and polyUb chains of different lengths and linkages to proteins plays a dominant role in very different regulatory mechanisms. Therefore, the study of polyUb chains has assumed a central interest in Biochemistry and Structural Biology. An essential step necessary to allow in vitro biochemical and structural studies of polyUbs is the production of their chains in high quantities and purity. This not always easy task can be achieved both enzymatically and chemically. Previous reviews have covered chemical cross-linking exhaustively. In this review, we concentrate on the different approaches developed so far for the enzymatic production of different Ub chains. These strategies permit a certain flexibility in the production of chains with various linkages and lengths. We critically describe the available methods and comment on advantages and limitations. It is clear that the field is mature to study most of the possible links but some more work needs to be done to complete the work and to exploit the current methodologies for understanding in full the Ub code.
Available from: P. John Hart
- "We also investigated the type of polyubiquitin linkage generated in the in vitro reactions by western blot using K63-and K48-linkage-specific antibodies (Figure 1C, left panel and right panel, respectively). Polyubiquitin chains synthesized by TRIM5a RING 1–93 and UbcH5c can be visualized by both antibodies because UbcH5c is a promiscuous E2 protein that generates both K48 and K63 linkages in vitro (Brzovic and Klevit, 2006; Kirkpatrick et al., 2006). In contrast, polyubiquitin synthesized by TRIM5a RING 1–93 and Ubc13 appears to be strictly K63 linked as is the case for the Ubc13/Mms2 control. "
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ABSTRACT: Members of the tripartite motif (TRIM) protein family of RING E3 ubiquitin (Ub) ligases promote innate immune responses by catalyzing synthesis of polyubiquitin chains linked through lysine 63 (K63). Here, we investigate the mechanism by which the TRIM5α retroviral restriction factor activates Ubc13, the K63-linkage-specific E2. Structural, biochemical, and functional characterization of the TRIM5α:Ubc13-Ub interactions reveals that activation of the Ubc13-Ub conjugate requires dimerization of the TRIM5α RING domain. Our data explain how higher-order oligomerization of TRIM5α, which is promoted by the interaction with the retroviral capsid, enhances the E3 Ub ligase activity of TRIM5α and contributes to its antiretroviral function. This E3 mechanism, in which RING dimerization is transient and depends on the interaction of the TRIM protein with the ligand, is likely to be conserved in many members of the TRIM family and may have evolved to facilitate recognition of repetitive epitope patterns associated with infection.
Available from: PubMed Central
- "Low concentrations of Ube2C formed heterotypic chains predominantly containing K11/48 linkages, but at high concentrations Ube2C formed complex chains with six different Ub linkages. This ability of the APC/ C to form multiple Ub linkages also has been observed in Xenopus extracts, where APC/C in combination with high concentrations of the E2 UBC4 (up to 4 mM) formed polyUb chains on cyclin B1 containing K11, K48, and K63 linkages (Kirkpatrick et al., 2006). Whether this degree of complex Ub-chain formation is physiologically relevant, or simply due to the high concentrations of E2s used in the in vitro assays, remains to be determined. "
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ABSTRACT: Proteasome-mediated degradation occurs with proteins principally modified with lysine-48 polyubiquitin chains. Whether the proteasome also can bind atypical ubiquitin chains, including those linked by lysine-11, has not been well established. This is critically important, as lysine-11 polyubiquitination has been implicated in both proteasome-mediated degradation and non-degradative outcomes. Here we demonstrate that pure homotypic lysine-11-linked chains do not bind strongly to the mammalian proteasome. By contrast, heterotypic polyubiquitin chains, containing lysine-11 and lysine-48 linkages, not only bind to the proteasome but also stimulate the proteasomal degradation of the cell-cycle regulator cyclin B1. Thus, while heterotypic lysine-11-linked chains facilitate proteasomal degradation, homotypic lysine-11 linkages adopt conformations that prevent association with the proteasome. Our data demonstrate the capacity of the proteasome to bind ubiquitin chains of distinct topology, with implications for the recognition and diverse biological functions of mixed ubiquitin chains.
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